Magnetic device for immunological analysis of a solid phase

ABSTRACT

An apparatus for performing immunoassays employs a microtiter tray having wells coated with a specific binding partner in conjunction with an array of magnets disposed below the tray. A device for providing relative circular motion of the tray and magnetic array so as to provide a magnet field to the well walls for attracting magnetic particles and subsequent movement of the magnetic field.

This application is a divisional of Ser. No. 07/916,494, filed Jul. 21,1992 now U.S. Pat. No. 5,318,914.

The present invention relates to a process for immunologicaldetermination or detection of biological substances, in particularantigens or antibodies, and devices for implementing this process.

Numerous techniques, of varying degrees of sensitivity and specificity,make it possible to detect the presence of antigens or the presence ofantibodies in biological media by formation of immunological type bondsbetween the substance to be determined and one or more known substances.

Some employ an immune adherence phenomenon, described in particular anAmer. J. of Clinical Pathology 82 (6)--719-721 (1984). In this method,the sample to be studied is introduced into a receptacle, generally thecup of a microtitration plate, on whose walls there are molecules orcells which have a specific affinity for the substance to be determined;a suspension of sensitized particles, that is to say on which there is asubstance capable of bonding by affinity with the substance to bedetermined, is also introduced and the distribution of these particlesafter a dynamic sedimentation phase is studied; in the presence of thesubstance being tested for, the particles evenly line the walls to whichthey are bonded via immunological type bonds occurring between thesubstance fixed onto the wall, that to be determined and the substancepresent on the particle; an even film is observable on the bottom of thecup; in the absence of the substance being tested for the particlescollect at the bottom, around the point of convergence of the walls, byforming a more opaque central spot. It is obvious that in the absence ofan external force applied to the particles, their sedimentation would betoo slow for the process to find application in routine fast biologicalanalysis and it is known that a suitable centrifugation makes itpossible to accelerate this type of sedimentation, but it requires theuse of bulky specific precision equipement which requires fineadjustment; as a matter of fact, on the one hand, the centrifugal forceapplied must be exactly parallel to the axis of symmetry of thereceptacles, to avoid having different distribution profiles accordingto the position of the receptacle relatively to the axis of rotationand, on the other hand, the centrifugation conditions must, in order toobtain good sensitivity and to avoid false results, be fixed for eachtype of substance to be determined and reactant since the duration andthe centrifugal force depend on them; furthermore, these techniquesemploying centrifugation, even if they are used for routine analyses, donot lead themselves to a compact, simple and cheap automation.

It has recently been proposed, in Patent Application WO 90/09590, toaccelerate sedimentation of the sensitized particles by the action of amagnetic force instead of a centrifugal force, by using sensitizedparticles containing a magnetic core--a process which overcomes thedrawbacks due to the use of a centrifuge.

Nevertheless, the Applicant has noticed, as will be seen further on,that this technique, in particular described in WO 90/09590, does notmake it possible to distinguish clearly reactions which are weaklypositive from reactions which are negative, so that it is not sensitiveenough for testing for irregular red blood cell antibodies and forantigens during pre-transfusion testing, or for lymphocytic antigensbefore tissue grafting.

The present invention provides a process which is distinctly moresensitive, specific and fast and can be partially or completelyautomated since the observation of the particles at the bottom of thereceptacles, whether they are transparent or opaque, can be carried outusing a photometer while the operations of filling the reactionreceptacles, incubating and washing can be carried out with conventionaldevices, which are known to the skilled man.

The subject matter of the present invention is a process for determiningor detecting by immuno adherence a biological substance present in asample, which consists in introducting the sample into a receptacle onwhose walls there is a component which has a specific immunologicalaffinity for the substance to be tested for, in adding magneticparticles on which there is a substance which has a specificimmunological affinity for the substance to be tested for, in subjectingthem to several successive magnetic actions in order to accelerate thedeposition of the said particles onto the walls and to displace thosewhich have not formed specific bonds with the substance to be tested forwhich has adhered to the walls via the component with affinity which isfixed thereto, and in observing the particles deposited.

In a first embodiment, the process according to the invention consists:

a) in introducing the said sample into a receptacle on whose walls thereis a component which has a specific immunological affinity for thesubstance to be tested for,

b) in adding magnetic particles on which there is a substance which hasa specific immunological affinity for the substance to be tested for,

c) in subjecting the particles to the action of a static magnetic fielsin order to coat the particles onto the walls, and thus to make possiblethe immunological reaction,

d) then in subjecting the receptacle to the action of a magnetic fieldof variable direction in order to displace and collect at the bottom theparticles which are not fixed by specific bonding to the walls via thesubstance to be tested for and the component with affinity which isfirmly fixed to the wall,

and finally,

e) in observing the particles deposited.

According to another aspect of this first embodiment, the particles arefurthermore subjected, before final observation, to a static magneticfield in order to remove from the receptacle the particles which do notadhere thereto via immunological bonds. For instance, it is possible tointroduce into the receptacle a suitably magnetized rod onto which onlythe particles which do not specifically adhere to the walls on thereceptacle will form a coat.

In a second embodiment, the process according to the invention consists:

a) in introducing the said sample into a recipient on whose walls thereis a component which has a specific immunological affinity for thesubstance to be tested for,

b) in adding magnetic particles on which there is a substance which hasan immunological affinity for the said substance to be tested for,

c) in subjecting the particles to the action of a static magnetic fieldin order to bring the particles onto the walls, and thus to makepossible the characteristic immunological reaction,

d) then in subjecting the receptacle to the action of a static magneticfield, for instance using a suitably magnetized rod, in order to removefrom the receptacle the particles which do not adhere to its walls by animmunological type bonding,

e) and finally in observing the particles deposited.

In a known manner, the said sample to be studied is in general left toincubate in the sensitized receptacle for a certain time, for example upto 30 minutes, at a temperature between approximately 20° C. and 45° C.before adding the magnetic particles.

A stage of washing between these first two stages of the process of theinvention is furthermore in general carried out, in particular when themagnetic particles are sensitized with a substance which would not havean affinity only for the substance to be tested for.

In general, routine immunological determinations are carried outsimultaneously in microtitration plates which comprise series ofcavities also called cups or wells, sometimes up to about a hundred,with a capacity of approximately 350 μl, arranged in parallel rows, orin cavities aligned on support bars, but any other receptacle ofsuitable dimensions could be used.

Another subject matter of the present invention is devices forimplementing the process according to the invention.

In what follows, the devices according to the invention are describedwhile referring to microtitration plates, but it is understood that theinvention is not limited to this type of receptacle, neither to anapparatus comprising a single plate. In these plates, the cups can beflat-bottomed, conical, frustoconical or hemispherical.

A device according the the invention, which is appropriate forimplementing the first embodiment of the process, comprises:

a support for keeping a microtitration plate horizontal, and capable ofgiving the whole of the plate horizontal circular translation movementwhose amplitude is between 1 mm and 15 mm in radius, as a function ofthe dimensions of the cups;

underneath, a set of magnets whose upper poles are parallel to the plateand are either arranged under each cup in a chequered pattern, possiblyalternately North and South, the space between them being of the orderof at least 2 mm, or advantageously arranged in a strip under each rowof cups, the poles of two neighbouring strips being alternately Northand South, and the space between two strips being very small, of theorder of 1/10 mm or less, it even being possible for the strips to becontiguous.

By circular translation, a movement is understood during which thecentre of the receptacle describes a circular path but carries out norotation around its axis.

According to an alternative embodiment, the device can comprise astationary plate support, and a set of magnets capable of carrying out ahorizontal circular translation movement.

Another device according to the invention, which is appropriate forimplementing the variant of the first embodiment or for implementing thesecond embodiment comprises further a series of magnetized cylindricalrods, of diameter less than that of the cups, and means for introducingthem simultaneously into all the cups, along their axis, as far as asmall distance from the bottom, for example between 0.1 and 1.5 mm, andremoving them therefrom.

These rods can be made of soft iron or steel, treated in order to makethem resistant to attack by the reaction medium; they are then duringtheir use in magnetic contact with a permanent magnet, which magnetizesthem. It is possible to have a single magnet for all the rods, or asmall magnet for each one. Furthermore, each rod can itself be apermanent magnet. The shape of the lower end of the rods is, preferably,adapted to that of the receptacle into which they areintroduced--hemispherical, conical or flat, accordingly--but it is alsopossible, in particular, to use a conical end in a hemispherical cup.

The rods, whose length can be greater than the depth of the cups andwhich can be wider at the top, are supported by a grating or a plate, asis optionally the case for the permanent magnets.

For implementing the second embodiment of the process, the set ofalternate magnets of the first device can be replaced by any means whichmakes it possible to create, in the zone where the receptacles are, amagnetic field, which will attract the particles towards the walls, suchas a uniformly magnetized plate; furthermore, in this case, themicrotitration plate support and the magnets are fixed with respect toeach other.

The receptacles will be able to be filled and washed manually or usingknown mechanical means in appropriate work-stations and it will bepossible to combine, with the magnetic treatment means according to theinvention, conventional means for filling and washing the microtitrationplates.

Furthermore, it will be possible to carry out observation of thedistribution profiles of the particles with the naked eye, preferablyusing a magnifying device, such as a binocular magnifying glass, and forvery high sensitivities with a suitable photometer coupled to arecording system; in the latter case, as is known, a transparentmicrotitration plate is used.

In the process according to the invention, the substance to be testedfor can be an antigen which is free or bound to the surface of a cellsuch as a virus, a lymphocyte, a platelet, an erythrocyte or anantibody, directed against a cellular or tissue antigen or against anallergen, presend in serum or in another biological medium.

The substances which have an affinity for the substance to be testedfor, that is to say which are capable of forming immunological typebonds with it, are then respectively antibodies or antigens. The capturecomponent, which is immobilised on the walls of the titrationreceptacle, will have to bind specifically the substance to bedetermined, whereas the revealing substance, which is on the magneticparticles, could be non specific. Numerous substances with suitableaffinity, currently used in immunological analysis, are known, such aspolyclonal or monoclonal antibodies, antiglobulins, lectins, red bloodcells and vital proteins, and the skilled mand will choose the one whichis suitable to the desired determination, without difficulty.

The component fixed on the walls can be an antibody or an antigen, whichare free or contained in the membrane of an intact cell or of a stromaor which come from a cell lysate.

The capture component is fixed onto the walls of the cups by techniqueswhich are well known to the specialists, either by absorption, when thecups are made of polystyrene, polyvinyl chloride or polymethacrylate, bycovalent chemical bonding with a compound contained in the material ofthe plate or deposited on the walls of the cups in an adherent film orby any other means known to the specialist, such as the one mentioned inEP-A-0350233 according to which red blood cells are bound via ionicdyes.

On the market there are a number of microtitration plates, said to besensitized, on whose cups there is the capture component or which areready to be sensitized by known techniques. The specialist knows thatnot all plates are suitable for determinations of the type of those ofthe invention, by immuno adherence; it is obviously desirable, in orderthat there should not be false positive results or negative results,that the density of the nodding sites is even and such that, therevealing particles are arranged on the walls in a single layer,preferably essentially continuous.

The magnetic particles will be of small size, of the order of 0.5 μm to5 μm and preferably between 0.8 μm and 1.7 μm and of a fairly homogenousparticle size, in general, consisting of a natural or synthetic polymer,which does not spontaneously adhere strongly to the material of thecavities; the polymer can coat a magnetic core or magnetic componentssuch as iron, ferrites and alloys which are dispersed in the polymerbulk. Suitable particles are commercially available, and those of thetrade mark Dynabeads® of the company Dynal or those of the trade markEstapor® of the company Rhone-Poulenc, which are composed of apolystyrene latex, containing up to 40% by weight of ferrite, may becited. Coloured particles can also be used.

The substance which is on the magnetic particles must have an affinityfor the substance to be determined; thus a species-specificanti-immunoglobulin for determining an antibody, or a polyclonal or amonoclonal antibody for determining an antigen, are suitable. They willbe fixed on the particle by one of the techniques known to the manskilled in the art, which are similar to those used for fixing on thecup wall; several molecules are in general fixed simultaneously on thesame particle, which makes it possible, if the local density of thesubstances to be determined which are immobilized on the walls issuitable, to obtain excellent adhesion of the particle.

In the first magnetic treatment phase, the titration cups are places instatic magnetic fields, whose distribution and intensity are identicalfor all the cups, and which are directed so that the particles are bringdownwards so that they deposit onto the surface of the walls. Themagnetic force must be sufficiently intense for the sedimentation to befast without being excessive, in order to avoid a collection of all theparticles at the lowest point, due to their pulling from the walls wherethey had previously been deposited.

In this phase, when the particles arrive near the sensitized walls,affinity bonds can be created between the revealing substance which ison the particle and the substance to be determined, which is immobilizedon the wall via the specific capture component.

In embodiments of the process in which the particles are subjected tothe action of a magnetic field of variable direction, during this phasethe particles which are weakly, or not, bonded ace pulled out and slidealong the walls so that they collect in the lowest zone of the cups.

A preferred means, according to the invention, for varying thisdirection consists, as previously mentioned, in subjecting themicrotitration plate to circular translation movements of small diameterabove the set of magnets so that the axis of a cup describes a circlewhose centre is located at the intersection of the line joining its axisto the one of a contiguous cup, which is located above a magnet of theopposite polarisation to the one underneath which the cup is placed,with the line of the air-gap between these two magnets; the radius ofthe said circle is less than half the width of a magnet, preferablybetween 10 and 40% of this width.

Under these conditions, all the points at the bottom of each receptacleare at one moment above the line of the air-gap, where the magneticfield is at a maximum, or in its vicinity, so that all the particleswhich are weakly bound are pulled off the walls in a very short time, inless than 5 minutes and even better, in most cases in approximately 2minutes.

The intensity of the magnetic fields to be applied depends in particularon the size and the density of the particles and on their concentration,on the viscosity of the reaction media, on the intensity of the forcesof affinity bonding and on the geometry of the cups, and the skilled manwill determine, by prior testing, their value and the suitable durationsof the various magnetic treatment phases which depend on them, in orderto obtain optimum sensitivity and the absence of false positive andnegative results for a reasonable treatment time.

Preferably, the bottom of the cups will be placed at a small distanceabove the set of magnets, which will make possible the use of magnets oflow power, in particular consisting of polymer strips containingmagnetized particles, which are found in the trade; this distance willbe, in general, of the order of 1 mm, from 0.1 to 5 mm, for magneticinductions less than 0.5 tesla.

For assays of antigens or antibodies in currently used microtitrationplates, of 12.8 cm×8.6 cm comprising 96 cups arranged in 12×8 parallelrows, with magnetic particles of diameter between 0.5 μm and 5 μm and adensity less than or equal to approximately 2, the average intensitiesof magnetic induction under the bottom of the cups are 0.2 to 0.4 tesla,whereas the dipping rods have a diameter of 3 mm, a length of at least10 mm and they create in the vicinity of their pole piece a magneticinduction of 0.10 to 0.20 tesla. The duration of the first phase ofdeposition of the particles in the stationary fields is from a fewseconds to 5 minutes, whereas the one of displacement in the magneticfields is less than 5 minutes, about 2 minutes in general, for arotation speed between 0.5 and 4 revolutions per second; finally, theduration of action of the magnetized rods will be in general from 5seconds so several minutes, possibly in several successive dips.

The process of the invention, in its various aspects, can be carried ourmanually, with simplified magnetic treatment equipment, but it can alsobe carried out in an automatic biological analysis apparatus whichcomprises, further to the components conventionally present in this typeof immuno assay automatic apparatus, at least one magnetic treatmentdevice according to the present invention.

Thus, an automated unit for testing antigens or antibodies by the immunoadherence process of the invention comprises a sample loader, awork-station for filling the microtitration plate cups with the samplesand the reactants, comprising the magnetic particles in suspension, amincubating-station, a washing-station, as well as a work-station forperforming the magnetic treatments according to the invention and awork-station for photometric reading wherein the observation, whetherquantitative or not, of the particles deposited in the cups, is done,the various work-stations being under the control of a microcomputer incharge of the various stages of the assay and its interpretation.

In order to make the invention better understood, particular embodimentswill be described with reference to the attached figures in which:

FIG. 1 represents a devide according to the invention in a plan view,with partial cutaway,

FIG. 2 represents, in a section along 2--2 and in a blown-up view, apart of the device showing the microtitration plate and the magnets,during the initial static magnetic treatment,

FIG. 3 represents, in a section along 3--3, the device according to FIG.1,

FIG. 4 represents, in section and in a blown up view, a part of a deviceused for implementing one variant.

FIGS. 1 to 3 represent a device for implementing the process accordingto the invention according to its first embodiment, comprising magnetictreatment means for carrying out determinations by immune adherenceusing microtitration plates.

The device represented in FIGS. 1 to 3 comprises a microtitration plate1 comprising a set of cups 2. This plate is fixed horizontally on thetwo parts of a support 3, which is itself fixed onto a plate 4 arrangedhorizontally which will be described in more detail further on withreference to FIG. 3.

As represented in FIG. 2, beneath the microtitration plate 1, at a smalldistance from and parallel to this plate, is a set 5 of magnets 6 in theshape of strips arranged under each row of cups 2 and whose upper polesare alternatively North 6a and South 6b, a very small space 7 (air-gap)being left between two strips.

The static magnetic field prevailing above the magnets 6 is representedsymbolically at 8.

The set 5 of magnets 6 is fixed to a parallelepipedal frame 9 underwhich a motor 11 is fixed.

The motor 11 drives a central cog wheel 12 via an axle 13. The motor cogwheel engages the lateral cog wheels 14, 15, on each of which there is asmall eccentric axle 16, 17 serving as a hub for the ballbearings 18,19.

These bearings are set in the plate 4 which will therefore be driven ina circular translation movement when the cog wheels carrying the smalleccentric axles 16, 17 rotate.

The cog wheels 12, 14, 15 are mounted on the lower plate 21 of the frame9.

In order to apply the static magnetic field the arrangement of themagnets as represented in FIG. 1 is used. The static magnetic fieldmakes it possible to bring the particles which are introduced into thecups 2 upon the walls.

In order to apply the magnetic field of variable direction, the motor 11is started rotating and the titration plate 1 which is fixed to theplate 4 is subjected to circular translation movements.

These circular translation movements of the cups 2 above the magneticfields make it possible to obtain within the cups 2, not only a magneticfield of variable direction, but also a slight agitation of the contentsof the cups 2, which contributes to the separation of the particleswhich are not bound or are weakly bound.

Preferably, before carrying out the circular translation movement, themicrotitration plate 1 is displaced with respect to the system ofmagnets, parallel to a row of cups, in the direction of alternation ofthe poles of magnets 6a and 6b, over a length approximately equal tohalf the distance between the axis of the cups; by this means, the axisof each receptacle is, during the rotations, nearer the zone of theair-gaps 7, where the magnetic field is at a maximum. The support isobviously provided with a means which makes this short translationpossible.

In a particularly preferred embodiment, the axis of each receptacle ispositioned at the beginning above the air-gap 7.

In FIG. 4, a device is represented which is appropriate for implementingthe variant of the first embodiment of the process or for implementingthe second embodiment of the process.

The device represented in FIG. 4 comprises a set of magnetized cods 22arranged in the same arrangement as the cups 2. Each rod 22 is made ofsoft iron or steel; it is of cylindrical shape; it has a hemisphericallower end 23 and an upper end 24 in contact with a permanent magnet 25.

This device is intended to be introduced into the cups 2 of themicrotitration plate after this plate has been subjected to a staticand/or rotating magnetic field and when each cup is still filled withthe reaction medium. The magnetized rods 22 are lowered using meanswhich are not shown and introduced into the cups 2 until the lower ends23 of the rods are a short distance from the bottom of the cups. Theadjustment of the distance and the centring of the rods 22 are ensuredby conventional means which are not shown.

The magnetized rods exert an attractive force in a direction which issubstantially opposite to the one of the first (static) magnetic fieldon the deposited particles, but only those which are not fixed or areweakly fixed by immunological bonds onto the walls of the cups 2 will beremoved.

In what follows, a description is given of examples of implementing theprocess according to the invention, using the devices for magnetictreatment of a microtitration plate which were described previously, fordetection of red blood cell irregular antibodies in serum, as well as,for comparison, the results obtained during a magnetic sedimentationunder the action of a constant field, as described in Patent ApplicationWO 90/09590, previously cited, or with the test marketed by the companyImmucor Inc.--Norcross--U.S.A. under the trade mark Capture R® whichinvolves a centrifugation.

This latter test for detection of irregular antibodies is carried out byan immuno adherence technique, in plates to which there have been fixed"test" (reference) red cells which are known to bear the antigenscorresponding to the antibodies to be detected. This process involves:

distributing the samples to be tested and the controls in the sensitizedcavities,

a fairly long incubation of approximately 20 minutes,

6 successive washings with an isotonic solution in order to eliminateall undesirable substances,

addition of revealing red blood cells, that is to say onto whichanti-human immunoglobulin antibodies have been fixed,

a centrifugation, in a centrifuge for a microtitration plate withcavities, lasting one minute at 1000 g, then observing the distributionprofiles of the red blood cells at the bottom of the cups; all theoperations carried out manually for a complete plate requireapproximately 1 hour.

The process according to the invention is distinctly more sensitive thanthese two known techniques.

EXAMPLE 1 Determination of an Irregular Red Blood Cells Antibody

a) Preparation of the magnetic particles coated with anti-humanglobulin.

Brown, latex magnetic particles of the trade mark Estapor® marketed byRhone-Poulenc (particle size 0.8 um) are washed with an aqueous solutionof KOH (pH 9-10) then suspended at a concentration of 1.2% (w/V) in aGBS buffer, composed of 0.1M glycine and 0.14M NaCl and adjusted to pH8.2 by adding NaOH.

A solution of an anti-human IgG antibody of concentration 0.2 mg/ml, isfurthermore prepared, in the same buffer.

The solution and the suspension are mixed in equal volumes and kept for45 minutes, under agitation at 37° C.

The sensitized particles are then separated and washed copiously withthe GBS buffer containing 0.1% of bovine serum albumin.

b) Preparation of the antibody solutions to be studied.

Dilutions are prepared, in a geometric progression of common ratio 2, ofa stock solution containing 1% (w/V) of an anti-D antibody, in anaqueous solution of NaCl (0.9%-w/V) containing bovine serum albumin(6%-w/V).

c) Determination of the antibodies.

In the assays which follow, a polystyrene microtitration plate 12.8cm×8.6 cm, whose 96 cups are U-shaped and are pretreated in order to fixred blood cells, is used; this plate is marketed by Immucor Inc. underthe trade mark Capture R®.

A suspension of washed reagent blood cells of concentration 0.5% (w/V)bearing the antigen corresponding to the antibody to be determined, isdistributed in the cups; the plate is centrifuged and the cups washedwith saline solution, then 50 ul of each dilution of the antibodysolution with 100 ul of the appropriate aqueous low ionic strengthsolution are introduced therein. In some of the cups, a negative controlis introduced which is constituted of inert human serum of group AB.

The plate is incubated for 20 minutes at 37° C. and then the cups arerepeatedly washed with an aqueous solution of NaCl (0.9%-w/V). Then 50μl of the previously prepared suspension of magnetic particles, broughtto a concentration of 0.02% in a GBS pH 8.2 buffer to which is added0.1% of bovine serum albumin, are introduced therein.

The plate is then put down onto the magnetic treatment devicerepresented in FIGS. 1 to 3, whose characteristics are the following:set of magnets formed of strips of Ferriflex® (rubber filled withmagnetic particles) marketed by the company DIC (France), of thickness 5mm, width 9 mm and length 70 mm, located less than 0.8 mm under thebottom of the cups, arranged in parallel rows and so that the axes ofthe cups are aligned on the middles of the strips or even better on theair-gaps.

The plate is left for 2 seconds on its support, then the support of theplate is driven for 2 minutes in a rotational movement whose radius is 3mm at a speed of approximately 50 revolutions per minute.

The distribution of the particles on the bottom of the cups is thenobserved with the naked eye: in the case of a negative reaction a largebrown pellet is observed in the middle of the bottom of the cup, whereasin the case of a positive reaction a brown film is observed over theentire bottom.

The negative control is further clearly distinguished from the cupscontaining anti-D at a dilution of 1/128 which was previously preparedfrom the 1% stock solution.

By way of comparison, an assay was carried out with a sensitizedmicroplate in the same way and with the same dilutions but using,instead of the sensitized magnetic particles, sensitized red blood cellsmarketed by the company Immucor for its Capture-R® test: the magnetictreatment phase was replaced by a centrifugation of the plate for 1 minat 1000 g, as recommended by this company; it was then possible todistinguish with the eye positive-result and negative result cups onlyup to a dilution of 1/32nd of the stock solution.

Sensitized plates were also prepared as previously, as well as 4.5 umDynabeads® magnetic particles marketed by Dynal under the reference M450, coated with anti-human immunoglobulin, applying the methoddescribed in Example 1 of Patent Application WO 90/09590 and 25 μl ofthese particles were introduced into the cups containing the anti-Ddilutions. The plate was then treated as described in Example 1 of thesaid application (homogenisation on a micro-agitator then action of astatic magnetic field). The last dilution giving a positive-resultdistribution which was barely observable with the naked eye is the oneof 1/16, whereas with the same particles subjected to the magnetictreatment described in the present example, a distinct distribution isobservable up to 1/32 with difference making interpretation very easy.

Finally sensitized Estapor® magnetic particles of 0.8 μum were used, andthey were subjected to the magnetic treatment described in WO 90/09590;the figures obtained were uninterpretable since the negative controlitself does not give the expected pellet at the bottom of the cup, evenif the duration of action of the magnetic field is extended up to 30minutes, whereas the same particles, subjected to the magnetic treatmentof the invention, give difference up to 1/128.

EXAMPLE 2

The microplates are prepared as in Example 1 but the magnetic treatmentis carried out in three steps, the first two steps being identical tothose of the preceding example; during the third, for two minutes,magnetized rods are previously described, of diameter 3 mm, areintroduced into each cup. After withdrawal of the rods, apositive-result distribution is then distinguished with a binocularmagnifying glass up to a dilution of 1/1024th of the 1% stock solution.

EXAMPLE 3

The magnetic treatment of the prepared plates as in Example 1 is such asto perform only the first and the third steps of the treatment describedin Example 2.

Observation with a suitable photometer allows the presence of apositive-result distribution to be noticed up to a dilution of 1/256thof the 1% stock dilution.

EXAMPLE 4 Reverse ANO Grouping, So-Called Simonin Test

A suspension of 0.02% (w/V) of Estapor® 0.8 μm magnetic particles coatedwith anti-human globulins is prepared, as well as microtitration platescoated with capture red blood cells as in Example 1 but of groups A1, A2and B. 50 μl of plasma to be studied and 10 μl of low ionic strengthsolution are introduced into each cup and incubation is carried out for20 minutes at 37° C. The cups are then washed and 50 μl of suspension ofmagnetic particles are introduced therein, before subjecting the plateto a magnetic treatment in two steps as in Example 2. Observation of thedistributions obtained allowed determination, without error, of thegroup A, B, AB or O of the plasma studied.

EXAMPLE 5 Forward ABO Grouping, So-Called Beth Vincent Test

In this assay, a polystyrene microtitration plate is used whose U-shapedcups are pretreated in order for them to be able to fix red blood cells,by the method described in Methods of Enzymology. vol 73, CH Heusser, JWStocke, RH Gisler, Academic Press Inc. (1981)

A suspension is prepared, as in Example 1, or 0.02% (w/v) of Estapor®magnetic particles, of 0.8% um diameter, but coated with anti-murineglobulins.

50 μl of a 1% (w/v) suspension of the red blood cells to be studied,which have been previously washed, are distributed in 3 cups per sampleand the plate is left for 5 minutes at room temperature before washingthe cups with saline solution. Into one of the three cups of eachsample, 50 μl of murine anti-A est serum and 100 μl of low ionicstrength solution are introduced; into another, 50 μl of murine anti-Btest serum and the same quantity of low ionic strength solution areintroduced, and into the third cup, anti-A+B test serum as well as a lowionic strength solution are introduced. After 20 minutes of incubationat 37° C., the cups are washed and 50 μl suspension of magneticparticles is introduced into each one, before subjecting the plate to amagnetic treatment in three steps as in Example 2.

Observation of the distribution of the magnetic particles made itpossible to determine without error the group A, B, AB or O of each ofthe 70 samples, of which some had a weak antigenicity, being groups A₃,A₃ B, Ax, B₃ or Bh.

EXAMPLE 6 Determination of IgG-Type Immuno-Globulins

A suspension of magnetic particles sensitized with an anti-human IgGantibody is prepared as in Example 1.

Moreover, the 96 cups of a rigid polystyrene microtitration plate,marketed by the company Polylabo (France) under the reference M 24 A,are coated with a goat anti-human IgG antibody as follows: 100 μl of anaqueous solution with 2.2 mg/ml of anti-IgG are introduced and left toincubate for 1 hour at 37° C. and one night at 4° C. After eliminationof the liquid, the cavities are filled with GBS buffer containing 0.1%of bovine serum albumin, and it is left in contact for one night at 4°C., then it is washed copiously with an aqueous solution of NaCl at 0.9%(w/V). The samples to be studied, of concentration 1 μg/ml, are preparedby dissolving immunoglobulin IgG marketed by Sigma under the reference I4506 in an aqueous solution of NaCl (0.9%) containing 2% of serumalbumin; the negative control samples only contain the diluent. Intoeach cup of the plate, 100 μl of sample are introduced and left toincubate for 45 minutes at 37° C. before washing with a saline solution.

50 μl of the suspension of magnetic particles brought to a concentrationof 0.02% in a GBS buffer--pH 8.2, to which is added 0.1% of bovine serumalbumin, are introduced into each cup, and the plate is subjected to themagnetic treatment described in Example 2. Positive-result cups aredistinguished perfectly from negative-result cups with the naked eye.

EXAMPLE 7 Detection of Irregular Red Blood Cell Antibodies

The solutions of magnetic particles and the solutions of the serums orplasmas to be studied which contain irregular antibodies dilutions up to1/2048th) are prepared as in Example 1.

On the one hand 26 sera or plasmas containing irregular alloantibodiesof C, c, E, D, K, Kpb, Fya, Fyb, Jka, Jkb, S or Lub specificity and, onthe other hand, 170 so-called negative plasmas which are known tocontain no irregular antibody are studied.

The cups are pretreated as in Example 5, then 50 μl of a 1% (w/v)suspension of the reagent red blood cells, whose specificity withrespect to the antibody to be tested for is known, is introduced intothe cups, which are washed after standing 5 minutes at room temperature.

For the study of plasmas containing the irregular antibodies, a cup withreagent red blood cells on which there is the antigen corresponding tothe antibody being tested for, and a cup with reagent red blood cellswhich do not contain this antigen, are prepared for each example.

For the study of undiluted negative plasmas, one cup contains reagentred blood cells on which there is the whole range or blood-groupantigens.

Into the washed cups, 50 μl of the solution to be studied and 100 μl oflow ionic strength solution are introduced, and they are left toincubate for 20 minutes at 37° C. The cups are then washed before 50 μlof the suspension of magnetic particles is introduced therein and theplate is subjected to the treatment in three steps described in Example2, before observing the distribution of the particles either with theeye or with a photometer.

No false positive reaction was detected on the negative plasmas, whichshows the good specificity of the process.

The titers, that is to say the last dilution for which a positivereaction was observed, for the antibody samples studied, are given inTable I.

The determination of the titer on a serum containing anti-D antibody wasrepeated 10 times on several occasions; no significant difference forthe 1/128 titer was observed.

By way of comparison, a determination of the 26 irregular antibodysamples was carried out with the kit marketed under the Immucor trademark Capture R. It was noticed that the process according to theinvention gave, for 56% of the samples, a net increase in sensitivitycorresponding to a titer difference of 1 to 3.

                  TABLE 1                                                         ______________________________________                                        SAMPLE No.    SPECIFICITY   TITER                                             ______________________________________                                        1             anti-c                4                                         2             anti-c + E            2048                                      3             anti-D + C    (for D  128                                                                   (for E  32                                        4             anti-E                128                                       5             anti-Fya              64                                        6             anti-JKb              256                                       7             anti-Kell             64                                        8             anti-D                >2048                                     9             anti-Kpb              128                                       10            anti-Lub              32                                        11            anti-D                128                                       12            anti-S                16                                        13            anti-c + E            512                                       14            anti-E                256                                       15            anti-E                64                                        16            anti-c + Kell (for K  64                                                                    (for c  64                                        17            anti-Fya              4                                         18            anti-E + C.sup.w                                                                            (for E  32                                        19            anti-Fyb              16                                        20            anti-JKa              32                                        21            anti-E                8                                         22            anti-Kell             256                                       23            anti-Fya              256                                       24            anti-c                128                                       25            anti-D + Fya  (for D  64                                        26            anti-E + Kell (for K  64                                                                    (for E  1024                                      ______________________________________                                    

We claim:
 1. A device for use in an immunoassay, wherein a substancewhich specifically binds a biological substance to be assayed in asample, immobilized on magnetic particles, is reacted with the sample inwells of a microtitration plate coated with a component whichspecifically binds the biological substance, and the distribution of theparticles as an indication of the presence or absence of the biologicalsubstance is observed, said device comprising:a horizontalmicrotitration plate having wells arranged in a zone, each well having acoating that can specifically bind the biological substance, and havingsupporting means comprising means for causing the plate to executehorizontally a circular translation movement, a set of magnetspositioned under the well zone, each magnet having upper and lowervertically aligned poles, with the upper poles disposed in a planeparallel to the plate and arranged in a checkered pattern of alternatingpoles or lines of alternating poles, said magnets arrayed so as toprovide at least one magnetic field for attracting magnetic particles toat least a portion of a wall of each said well, when the supportingmeans is in a first position.
 2. The device according to claim 1 whereinsaid magnets comprise magnetized polymer.
 3. A device for use in animmunoassay, wherein a substance which specifically binds the biologicalsubstance to be assayed in a sample, immobilized on magnetic particles,is reacted with the sample in wells of a microtitration plate coatedwith a component which specifically binds the biological substance, andthe distribution of the particles as an indication of the presence orabsence of the biological substance is observed, said devicecomprising:a horizontal microtitration plate having wells arranged in azone, each well having a coating that can specifically bind thebiological substance, and having motionless supporting means, a set ofmagnets positioned under the well zone, each magnet having upper andlower vertically aligned poles, with the upper poles disposed in a planeparallel to the plate and arranged in a checkered pattern of alternatingpoles or lines of alternating poles, said magnets arrayed so as toprovide at least one magnetic field for attracting magnetic particles toat least a portion of a wall of each said well when the set of magnetsis in a first position, means for causing the set of magnets to executea horizontally relative circular translation movement with respect tothe supporting means.
 4. The device according to claim 3 wherein saidmagnets comprise magnetized polymer.